Method and Configuration for the Optical Detection of an Illuminated Specimen

ABSTRACT

A method for the optical detection of an illuminated specimen, wherein the illuminating light impinges in a spatially structured manner in at least one plane on the specimen and several images of the specimen are acquired by a detector in different positions of the structure on the specimen. An optical sectional image and/or an image with enhanced resolution is then calculated. The method includes generating a diffraction pattern in the direction of the specimen in or near the pupil of the objective lens or in a plane conjugate to the pupil. A phase plate with regions of varying phase delays is dedicated to the diffraction pattern in or near the pupil of the objective lens or in a plane conjugate to said pupil, and different phase angles of the illuminating light are set.

CROSS REFERENCE

This application is a continuation of, and claims priority benefit ofcopending U.S. patent application Ser. No. 14/967,088 filed on Aug. 14,2013, which is a divisional of, and claims priority benefit of, U.S.patent application Ser. No. 12/323,054, now U.S. Pat. No. 8,537,461,which claims the priority benefit of the filing date of U.S. provisionalpatent application 60/990,007 filed on Nov. 26, 2007, the contents ofall of which are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to the field of microscopy, andmore particularly to microscopy in which structured illumination is usedfor depth discrimination in the wide field and for enhancing resolutionand contrast.

BACKGROUND OF THE INVENTION

In microscopy, structured illumination is used for depth discriminationin the wide field [1]¹ and for enhancing the resolution and the contrast[2]. Generally, a grating or another periodic structure is projectedinto the specimen [3] or an interference pattern is generated in thespecimen by means of interference of coherent component beams [4].Subsequently, images that differ from one another due to the shift ofthe illumination structure are suitably blended with one another so asto obtain an optical sectional image or an image with enhanced contrastand enhanced resolution [5,6]. ¹ Bracketed references refer to the listof references at the end of the specification prior to the claims.

All of these methods have in common that different phase angles of theperiodic structure are projected into the specimen; at the same time, itis desirable that the phase angle can be controlled as accurately aspossible and that a rapid change between various settings of the phaseangle can be made. An inaccurate shift of the projected structure canlead to artifacts (e.g., striation) in the image. The accuracy, speed,reproducibility and calibratability of the setting of the phase angleare therefore very important characteristics for implementing such amethod for structured illumination.

In addition, in some approaches to the implementation of the method ofthe structured illumination, in particular in the high-resolutionapproach [7], a rotation of the orientation of the projected structureis required.

SUMMARY OF THE INVENTION

The invention relates to the implementation of a structured illuminationmethod. Compared to the prior art, the proposed configuration has beensimplified and can offer the advantage of making a high scanning speedpossible. In addition, in developing the invention, the precision of thephase shift of the periodic structure is no longer critically dependenton the precision of the final control elements. The precision ispredetermined solely by the quality of a phase element. This is asignificant advantage over the prior art and obviates the repeatedrecalibration of the configuration and can help reduce artifacts. Inaddition, the proposed configuration is able to operate with a widerange of wavelengths, and the phase shift as well as the orientation ofthe projected structure can be set requiring only a single mechanicalactuator.

In the introduction below, the underlying principles will be describedprior to providing detailed explanations of practical examples accordingto the present invention.

In the literature (identified in the list of references at the end ofthis specification and before the claims), a distinction is made betweentwo types of structured illumination, with the first type being used toenhance the lateral resolution and also requiring that the orientationof the illumination structure (e.g., a line structure) generated in thespecimen space be changed. The second type of structured illuminationcan be used to generate optical sections without leading to anenhancement of the lateral resolution (Zeiss APOTOM). The presentinvention can be used for both types of structured illumination or evenfor combinations of the two.

The configurations according to the present invention are based on theobservation that a periodic structure (for example, a grating) which islocated in a plane conjugate to the specimen and which generatesstructured illumination in the specimen generates a diffractiondistribution in the pupil of the objective lens. In addition, advantageis taken of the knowledge that the direct manipulation of thediffraction orders in the pupil determines the shape of the producedillumination structure in the specimen. Manipulating the diffractionorders can include: selecting or suppressing certain orders, rotatingthe diffraction orders in the pupil plane about the optical axis of theconfiguration, or setting the relative phase between the diffractionorders before they enter into the pupil of the objective lens used. Byselecting or rotating the diffraction orders in the pupil, it ispossible to influence the orientation of the illumination structure. Therelative phase angle between the diffraction orders controls the phaseangle of the structured illumination.

In a first practical example of the invention, a type of implementationwill be used, which, in addition to setting the phase angle, allows theorientation of the projected structure in the specimen to be changedand, within a certain range, is independent of the wavelength of thelight of the projected structure used. The property of wavelengthindependence and the possibility of setting the orientation are notrequired in all applications or implementations. The implementation ofthese additional properties is therefore optional. The inventioncomprises the combined implementation of all three properties (phaseshift, change in orientation, wavelength independence) but is notlimited to this combination.

In a first measure, a periodic structure is added in an intermediateimage plane conjugate to the specimen. Preferably, this is a periodicphase structure. The simplest embodiment can be a phase grating (linegrating); however, two-dimensional structures are also conceivable.

Below, the invention will be explained using an implementation whichmakes it possible to orient the illumination structure in N/2=3directions, and given a suitable design, another number of orientationsis possible as well. A two-dimensional grating, such as shown in FIG. 1,can be used as the periodic structure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an illumination structure grating disposed in anintermediate image plane conjugate to the specimen.

FIG. 2 illustrates a typical illumination distribution in the pupil ofthe objective lens or in a plane conjugate to said pupil.

FIG. 3 illustrates a diaphragm structure.

FIG. 4 shows a phase structure which is inserted into the upper openingof the diaphragm shown in FIG. 3.

FIG. 5 illustrates a mask with a phase structure.

FIGS. 6(a)-6(f) show the selection of the diffraction orders and thesetting of the relative phase angles between the diffraction orders.

FIG. 6(a) illustrates a first setting of the relative phase angles and afirst phase angle.

FIG. 6(b) illustrates a first setting of the relative phase angles and asecond phase angle.

FIG. 6(c) illustrates a first setting of the relative phase angles and athird phase angle.

FIG. 6(d) illustrates a second setting of the relative phase angles anda first phase angle.

FIG. 6(e) illustrates a second setting of the relative phase angles anda second phase angle.

FIG. 6(f) illustrates a second setting of the relative phase angles anda third phase angle.

FIG. 7 illustrates the diffraction distribution in the pupil generatedby the grating shown in FIG. 1.

FIG. 8 shows a modified phase plate which makes chromatic correctionpossible.

FIG. 9 shows the radial dependence of the phase mask for the chromaticcorrection of the configuration.

FIG. 10 shows a modified phase element.

FIG. 11 is a schematic diagram of a configuration of the presentinvention for fluorescence microscopy.

FIG. 12 illustrates a configuration in which the diaphragm and the phasemask are connected via a gear unit.

FIG. 13 shows the view of a plane perpendicular to the optical axis (OA)in which the outputs of fiber optics (FO) are held in place by means ofa ring-shaped fiber holder (FH).

FIG. 14 is a lateral view illustrating a fiber-based implementation ofstructured illumination.

FIG. 15 is a lateral view illustrating how an existing opticalmicroscope system can be modified.

FIG. 16(a) is a lateral view illustrating a micro-optical module.

FIG. 16(b) is a top view illustrating the micro-optical module of FIG.16(a) with a sightline perpendicular to the optical axis.

FIG. 17 illustrates another practical example of a configuration whichmakes the pupil accessible by way of an intermediate image.

FIGS. 18(a) and (b) illustrate two variants of an achromatic beamdivider.

FIGS. 19(a) and (b) illustrates a polarizer that can be fitted to arotating mask and

FIG. 20 shows the transmission through a polarizer in the mask as shownin FIG. 19.

DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

The invention will now be explained in detail based on practicalexamples.

FIG. 1 shows a structure (phase structure, honeycomb structure) that isdisposed in an intermediate image plane conjugate to the specimen, inthis case, with an N=6-fold symmetry. The circular ring marks theillumination.

The structure shown in FIG. 1 generates a diffraction pattern in thepupil, again with a 6-fold symmetry of the diffraction orders, withoppositely lying pairs of the illumination corresponding to theorientation of one structure in one spatial direction.

A typical illumination distribution in the pupil of the objective lensor in a plane conjugate to said pupil is shown in FIG. 2. The lightdistribution generated by the structure shown in FIG. 1 in the pupil isillustrated. The points P drawn in are the sites of the diffractionorders, and the ring R shows the boundary of the pupil. The 0^(th)diffraction order is preferably located on the optical axis OA. Given anN=6-fold symmetry, a rotation of the illumination pattern by φ=360/N=60degrees about the optical axis would reproduce the original structure.The broken lines connect the diffraction orders which lead to thestructure in one spatial direction.

Next, an additional element is disposed near the pupil or in a planeconjugate to said pupil. According to the present invention, thiselement comprises a diaphragm which will be described in greater detailbelow. The function of this diaphragm is the selection and/orsuppression of certain diffraction orders.

This type of diaphragm structure is shown in FIG. 3. It comprises twoopenings that are symmetrically opposite with respect to the opticalaxis, with the size of the angle segments occupied by these openingscorresponding approximately to the angle defined by the symmetry of thediffraction pattern

φ=360/N

or being slightly larger. In FIG. 3, the two openings are white, thecircle segments correspond to an angle of φ. The optical axis is locatedat the intersection of the axes of symmetry that are identified by thebroken lines.

A diaphragm structure can be combined with a phase structure and isdisposed in the pupil or in a plane conjugated to the pupil.

According to the present invention, the diaphragm, depending on thediaphragm setting, selectively transmits only certain diffraction ordersin the diffraction image in the pupil, which diffraction orders,relative to the optical axis, lie opposite each other. Thus, by rotatingthe diaphragm, specific diffraction orders can be selected, and theorientation of the illumination structure in the specimen can beinfluenced.

As an alternative to the use of the diaphragm structure shown in FIG. 3in combination with the two-dimensional grating shown in FIG. 1, it isalso possible to use a grating which is preferably structured only inone dimension (e.g., a line grating) and which generates a pair ofpoints (depending on the type of lattice, with or without the 0^(th)order) as in FIG. 2 in a position conjugate to the specimen, and torotate said grating about the optical axis. In comparison to thecombination of a 2-D grating and a diaphragm for selecting diffractionorders, this configuration has an increased light yield but, compared toone of the following practical examples, it also requires an additionalfinal control element to rotate the grating. The shift of the phaseangle also takes place as described below for the combination of the 2-Dgrating and the diaphragm.

Below, the manipulation of the relative phase of the diffraction orders,as it can be used to shift the periodic structure in the specimen, willbe explained.

According to the present invention, a structured phase plate is added tothe configuration. This phase plate can be disposed on a separateelement near the pupil or it can be inserted into one or both openingsof the diaphragm shown in FIG. 3. Without loss of generality, the use ofonly one phase plate will be described in this document, which phaseplate is inserted into the upper opening of the mask seen in FIG. 3 andwhich, in combination with the diaphragm, forms a functional unit. Suchan integrated combination of a phase plate and a diaphragm isillustrated in FIG. 4.

FIG. 4 shows a phase structure which is inserted into the upper openingof the diaphragm shown in FIG. 3. The different gray values of theequidistantly divided circular sectors correspond to different phasedelays with fixed, predefined steps. For a given wavelength λ, the phasedifference between two neighboring circular sectors is

σ=2π/P,

wherein the phase difference 2π corresponds to an optical pathdifference of λ.

At a minimum, the phase structure has three different phases whichcorrespond to three setting steps (without 0^(th) order). With the0^(th) order, which, through an opening in the center of the diaphragm(not shown in FIG. 3 or any relevant subsequent figures) on thespecimen, at least 5 phases are required.

FIG. 4 illustrates a phase element for P=5 phase steps, which phaseelement comprises 5 sectors. In a useful embodiment of the invention,the angle segments of the phase element are divided into angle segmentsmeasuring

γ=φ/P.

FIG. 5 shows the mask with the phase structure as it is disposed in thepupil. The mask and the phase structure are rigidly connected to eachother and disposed so as to be able to pivot about the optical axis.

FIG. 5 shows the configuration of the masks/phase structure in thepupil. The rim of the pupil is marked by a red circle. The sites of thediffraction sites in the pupil are marked in green as in FIG. 2.

By rotating the structure shown in FIG. 5 in the pupil plane, thediffraction orders in the pupil are selected. Only the oppositely lyingorders (symmetrical) pass the mask; all other orders impinge onnontransparent regions of the mask. This sets the orientation of thestructure generated in the specimen. In addition, by means of smallerrotational steps, an alternation between two circle segments of thephase element takes place. As a result, the relative phase difference ofthe [sic; word or words missing] through the two oppositely lyingopenings of the mask is reset. This functioning mode is illustrated inFIG. 6.

FIGS. 6(a)-6(f) show the selection of the diffraction orders and thesetting of the relative phase angles between the diffraction orders.

The line distribution generated in the specimen has an orientation 1 inthe component images a) to c) and an orientation 2 in the componentimages d) to f), since different pairs of diffraction orders can passthrough the mask. In addition, 3 different phase steps of the generatedstructure are shown since the diffraction orders transmitted through thetwo openings of the mask structure undergo different relative phaseshifts, depending on the sector of the phase plate in which thediffraction orders are located. To set the phase step, only smalladjustment steps are required, which makes it possible to reach a highspeed. In addition, by continuously rotating the element, it is possibleto pass through a sequence of phase steps and orientations of thestructure, and the angle position of the element can be synchronizedwith the image acquisition by utilizing an appropriate angle encoder.

It should be emphasized that the precision of the relative phase shiftdoes not depend significantly on the precision (angular resolution) ofthe shift of the phase plate and/or the mask. This is an importantadvantage over the prior art [8,9] since, according to the presentinvention, the precision of the relative phase steps now only depends onthe quality of the phase plate. As a result, a calibration is no longerrequired and a disadjustment is impossible. The only prerequisite isthat the angle be positioned in a manner to ensure that the twodiffraction orders are located in the correct sector of the phase plate.The adjustment precision and reproducibility required to position theangle are of comparably little importance. In a useful embodiment of theinvention, it is therefore possible to solve this problem through theuse of a conventional stepping motor. In contrast to the prior art[9,8], neither piezo actuators nor galvanometer scanners are required toshift the phase. This constitutes a considerable cost advantage.

The configuration according to the invention as explained above isdependent on the light wavelength used, and it is therefore preferableto use monochromatic light. According to the present invention, theproposed configuration can be modified in such a manner that it canoperate in a whole range of wavelengths. This chromatic correction willbe explained in greater detail below.

Looking at FIG. 7, we will first consider the effects of a secondwavelength (blue marked diffraction orders) in a blue shift relative tothe first wave length (green marked diffraction orders). Here, thesmaller wavelengths are less strongly diffracted and are thereforelocated at a shorter radial distance from the optical axis.

FIG. 7 illustrates the diffraction distribution in the pupil generatedby the grating shown in FIG. 1. The points closer to the axis are thesites of the diffraction orders of the longer wavelength λ₁, and thepoints located farther away mark the sites of the diffraction orders ofthe shorter wavelength λ₂. The ring marks the boundary of the pupil. The0^(th) diffraction order is preferably located on the optical axis OA.

A chromatic correction of the configuration can now be implemented by anadditional radial dependence of the phase delay of the phase plate shownin FIG. 4. This is illustrated in FIG. 8. The radial dependence isselected to ensure that, when switching to the next sector (by rotatingthe element), for each wavelength, the same correct phase jump, relativeto the wavelength, occurs.

FIG. 8 shows a modified phase plate which makes chromatic correctionpossible.

The diffraction orders of the wavelengths λ1 and λ2 are located atdifferent radial distances r1 and r2 from the optical axis within aplane conjugate to the pupil. As already mentioned earlier, the lightdistribution in the pupil is generated by the element illustrated inFIG. 1. Thus (see FIG. 8):

λ1>λ2 and λ1/λ2=r1/r2.

FIG. 8 illustrates the example of a phase plate with P=5 phase steps.Thus, for any wavelength λ, the diffraction order of which is located ata distance r, the optical path difference OPD generated by the phaseplate must be:

OPD=λ/P.

From this follows that the phase plate for chromatic correction musthave an optical path difference that must be proportional to the radiusr. According to the present invention, the radial dependence of thephase plate can be continuous or quasi-continuous. If a discrete numberof fixed wavelengths are used, the radial dependence of the phase platemay also be constant in certain sections.

An example of a chromatic correction of the phase plate for a randomdistribution of wavelengths is illustrated in FIG. 9. Here, only asingle sector of the phase mask is shown; the gray values represent theposition-dependent optical path length which can be implemented, e.g.,by variable plate thicknesses or suitable refractive index changes, andwhich is directly proportional to the radial distance r.

FIG. 9 shows the radial dependence of the phase mask for the chromaticcorrection of the configuration. The optical path length changes(preferably) linearly with the radius.

It should be emphasized that the chromatic correction as described aboveis a useful but by no means necessary feature of a configuration forstructured illumination. Using suitable calibration, thewavelength-dependent phase steps can be determined and properly factoredinto the algorithms.

For energy reason, among other things, it may be useful not to block allbut two diffraction orders in the pupil by means of a diaphragm asdescribed above. In this case, it is possible to allow simultaneousstructuring in all those spatial directions that are generated by theelement in the intermediate image plane by means of diffraction.Compared to the sequential acquisition of the images with differentstructure directions, the number of the images to be acquired atdifferent phase angles remains the same. The phase angles can now be setby exchanging the element shown in FIG. 5 for a completely transparent(phase) element as seen by way of an example in FIG. 10.

FIG. 10 shows a modified phase element in or near the pupil plane (or aplane conjugate to said pupil plane) for the above-mentionedsimultaneous structuring in several spatial directions. By rotating themask shown, the phase angles of the structure directions are set. Thenumber of phase steps is N/2*P. With the structure of FIG. 1 (N=6) andwith P=3, one obtains 9 phase steps. As described above, each segmentcan have a radial phase shape so as to allow equidistant phase steps fordifferent colors.

A typical configuration according to the present invention forfluorescence microscopy will be explained below based on FIG. 11. Thespecimen (1) is attached to a specimen holder (3) that can be positionedin front of the microscope objective lens (5). The elements with phasestructure (7) described and shown in FIGS. 5 and 10 are disposed nearthe pupil (9) of the microscope objective lens (5) or in a planeconjugate to said pupil. Also included is an area detector (11) (e.g., aCCD). The configuration for fluorescence detection described alsocomprises a main color divider (13), for separating fluorescent lightand excitation light, and an emission filter (15). The mask (7) can bemoved by means of an actuator (17), which can be a stepping motor torotate the mask or a liner drive. A stepping motor can drive a circularmask directly or indirectly via a gear unit or another mechanical unit.An additional mask (19) is disposed in an intermediate image plane whichis conjugate to the specimen plane (1) and which is generated by thebarrel lens (21). The light source (23) illuminates the element (19) andcan be, for example, a laser, an LED, a high-pressure mercury lamp or aconventional incandescent lamp. Also included can be an optionalcollimation optics system (25). The light source (23) can bemonochromatic or emit several wavelengths simultaneously orsequentially.

In a preferred embodiment of the present invention, the phase mask andthe diaphragm for making a selection are disposed in or near the pupilof the objective lens. However, due to design features, the pupil inmany microscope objective lenses is not directly accessible. In thiscase, a relay optics system can be used to generate an intermediateimage in a freely accessible intermediate image plane, in the vicinityof which the phase mask and the diaphragm can be disposed.

Depending on the distance of the phase mask from the pupil plane, thediameter of the individual diffraction orders (see FIG. 2) will increasewith the distance from the pupil plane. Thus, the extension of thediffraction orders in the tangential direction also defines and limitsthe minimum size of the sectors of the phase plate (see FIG. 5).According to the present invention, this problem can be solved with auseful configuration in which the diaphragm for selecting thediffraction orders and the phase element are located on two differentelements that are rigidly connected to each other, with both elementsbeing disposed so as to be able to pivot about the optical axis, with agear unit coupling the two elements and defining the ratio between therotational speeds. This type of configuration is illustrated in FIG. 12,which shows a lateral view perpendicular to the optical axis. Thisconfiguration has the advantage that a diaphragm with a relatively smallopening can be combined with a phase element with relatively largeangular segments and that to move the two elements, only a singleactuator (e.g., a stepping motor) is required. The chromatic correctionin the radial direction is also influenced by the size of thediffraction orders in the plane of the mask, which must be factored induring the construction of the mask. It is therefore generally useful toposition the mask as far as possible in the pupil plane to minimizethese effects.

FIG. 12 shows a configuration in which the diaphragm and the phase maskare connected via a gear unit. The entire configuration is disposed inor near the pupil of the objective lens. The diaphragm for selecting thediffraction order (left in FIG. 12) can have a form as illustrated inFIG. 3. The phase mask (right in FIG. 12) can have the form asillustrated in FIG. 10. The gear unit connects the two elements and canbe located near the optical axis or at the rim of the diaphragm and/ormask. The mounting support for the diaphragm and/or mask can itself beconstructed as a gear wheel which is disposed so as to pivot about theoptical axis.

According to another embodiment of the present invention, the lightdistribution in the pupil can be generated directly by positioningoptical fibers in the pupil of the objective lens instead of having itbe diffracted by a periodic structure. This type of configuration isshown in FIG. 13. As can be seen, n outputs (in this case, n=6) of thefiber optics (FO) are disposed near the pupil, which outputs are held inposition by a fiber holder (FH). The optical axis (OA) is disposedperpendicular to the thawing plane and (PB) marks the pupillaryboundary. Each of the outputs of the fiber optics (FO) can comprise amicro-optical unit, for example a microlens, a structured surface or aspecial coating.

FIG. 13 shows the view of a plane perpendicular to the optical axis (OA)in which the outputs of fiber optics (FO) are held in place by means ofa ring-shaped fiber holder (FH). (PB) marks the boundary of the pupil.

The configuration of fibers in the pupil has the advantage that theorientation of the illumination structure in the specimen can bedetermined very rapidly by switching the light to certain fiber outputs.This can be accomplished, for example, by electronically switching lightsources which are dedicated to the individual fiber outputs. Inaddition, the fiber optic switch (FSE) can be designed to ensure thatthe light can be rapidly switched between the individual fiber outputs(by switchable light coupling or blocking).

FIG. 14 shows a lateral view to illustrate the fiber-basedimplementation of structured illumination: The optical axis (OA) of theobjective lens (0) passes through the pupil (P) of the objective lens.The fiber optics (FO) are placed near the pupillary boundary (PB) andheld in position by means of the fiber holder (FH). In thisillustration, only two fiber optics are shown, but it is also possibleto use a larger number. The fiber optic switching unit (FSE) can switchlight onto certain fibers and also set the relative phase of the lightemitted by the fiber optics.

Such a configuration for structured illumination using an optical fiberimplementation is shown in FIG. 14. The Figure shows a lateral view inwhich the optical axis is disposed in the drawing plane. The outputs ofthe fiber optics (FO) are disposed near the pupil (P) of the objectivelens (O), with (PB) marking the boundary of the pupil. To simplify thedrawing, only two fibers are shown in the figure, but a larger numbercan be used. The optical fiber switching unit (FSE) has severalfunctions: it contains a light source and/or possibilities formanipulating the spectral composition of the light used. In addition,the unit (FSE) can selectively distribute the light to a combination ofcertain fibers and also control the relative phase angle of the lightbetween the active outputs of the fiber optics (FO).

As already mentioned, the pupil of the objective lens is not alwaysdirectly accessible. Therefore, regardless of the implementation (e.g.,fiber configuration or use of phase plates), it may be necessary to makethe pupil accessible by means of an intermediate image. Intermediateimages of this type are known from the prior art and can be implementedby means of intermediate optics systems. For the sake of clarity, suchintermediate optics systems have been omitted in the drawingaccompanying this document. However, these intermediate optics systemscan be used if required by the conditions of the optical configuration.

Yet another practical example according to the present invention uses acombination of optical fibers and a micro-optics system to implement thedescribed method for the structured illumination and to generate therequired illumination distributions in the pupil. Especially inmicroscope objective lenses, the plane of the pupil of the objectivelens in most cases is located within the objective lens, but can be madeaccessible by means of an intermediate image. The configurationdescribed solves the problem of generating the illumination distributionand the intermediate image in an especially compact manner and istherefore especially suitable for integration into existing microscopicsystems. FIG. 15 shows the lateral view of a microscope objective lens(O) with a lateral slide-in compartment (ES) which is often used to holdDIC (differential interference contrast) prisms. The mechanical slide-incompartment (ES) inside the objective lens holder (OBA) is often anintegral part of the lens turret.

FIG. 15 shows a lateral view to illustrate how an existing opticalmicroscope system can be modified. Optical axis (OA), objective lens(0), specimen (PR), objective lens holder (OBA), slide-in compartment(ES).

Next, a component will be described which is preferably disposed in thearea of the slide-in compartment (ES) in FIG. 15 and which solves theproblem of generating illumination distributions according to thepresent invention.

FIG. 16 shows a micro-optical module (MOM) from a lateral view a) andfrom a top view with a sightline perpendicular to the optical axis b).The micro-optical module (MOM) is preferably connected to the fiberoptics (FO) and can comprise a combination of the followingmicro-optical components: fiber optic light guide, optical beamdividers, micro-optically integrated mirrors, microlenses.

In the illustration according to FIG. 16, the fiber optics (F0) areconnected to the light guide channels (LLK). The entire module (MOM) canbe inserted into the slide-in compartment (ES), with the upper surfaceof the module facing the objective lens. (OA) marks the optical axis ofthe objective lens. Micro-optically integrated mirrors (MSP) can be usedto deflect the light fed in by way of the fiber optics (FO). Themicrolenses (MIL) emit the fed-in light and preferably focus it into theplane of the pupil of the objective lens (not shown).

FIG. 16 serves to illustrate a micro-optical module (MOM). View a) is alateral view perpendicular to the optical axis (OA) of the microscopeobjective lens (FIG. 15), and b) is a top view of the module. The entireassembly can be inserted into the slide-in compartment (ES) shown inFIG. 15. The axis marked by (OA) is the axis in the inserted state ofthe compartment and is defined in FIG. 15. The fiber optics (FO) areconnected to the light guide channels (LLK). Micro-optically integratedmirrors (MSP), microlenses (MIL) and a preferably circular transparentregion (TRB).

Although, for clarity's sake, the light guide channels (LLK) in FIG. 16a) are shown one on top of the other, it is preferable to dispose themin a single plane. The transparent region (TRB) is transparent to thelight to be detected, which light comes from the objective lens. In FIG.16, this region is smaller than the circular region that is defined bythe microlenses (MIL). The light coming from the specimen will fill atleast this region. It may therefore be useful to lay out this region ina size large enough to correspond to the maximum illuminated region inthis area. Since the microlenses (MIL) and all channels (LLK) causelosses, a construction optimized for transmission (e.g., by having thechannels serve as waveguides) is to be preferred. Preferably, it isimplemented in the form of a recess in the substrate of themicro-optical module (MOM). It is useful to dedicate exactly one fiberoptics (FO) to each microlens (MIL). In FIG. 16 b), the light guidechannels (LLK) which form the connection between the fiber optics (FO)and the microlenses (MIL) as well as the micro-optically integratedmirrors (MSP) are not shown in detail but only schematically by means oflines (LLK). The fiber optics (FO) are preferably connected to a fiberoptic switching unit (FSE) as shown in FIG. 14 and explained in the textassociated with this figure. This fiber optic switching unit makes itpossible to switch the light in the individual fiber optics (FO) in atargeted manner; in addition, it is possible to set the relativeintensity in the fiber optics as well as the relative phase of the lightin the individual channels. This type of fiber optic switching unit canbe based on piezo actuators. To generate point-shaped lightdistributions at the site of the plane of the pupil of the objectivelens, the focal length of the microlenses (MIL) used can beappropriately adjusted.

Yet another practical example will describe a configuration which makesthe pupil accessible by way of an intermediate image, on the one hand,and which makes it possible to use an achromatic color divider with highefficiency (as described in [10]), on the other hand. An opticalconfiguration of this type is shown in FIG. 17. The periodic structure(PS), which can have the form shown in FIG. 1, is illuminated, and thediffraction image is imaged by means of optical elements into anintermediate plane (ZE) which is conjugate to the pupil of the objectivelens and in which the phase element (PE) is located. The diffractionorders of the periodic structure are preferably focused into theintermediate plane (ZE). The phase element (PE) can have the form shownin FIGS. 4, 5 and 8 and is preferably disposed so as to be able to move.As described earlier, the function of the phase element is to adjust therelative phase angle of the diffractions orders relative to one another.By means of an additional intermediate optical system, the intermediateplane (ZE) is imaged onto the beam divider (ACG). This element (ACG)comprises a transparent substrate with one or more reflecting regions asshown in FIG. 18. The principle of the achromatic beam divider isdescribed in [10]. Details will be discussed in a practical examplebelow. The beam divider (ACG) feeds the excitation light (AL) to theobjective lens (O). The detected light (DL), which is preferablyfluorescent light, is transmitted through the transparent regions of thebeam divider and is fed to an associated detection unit which cancontain the area receiver. The achromatic beam divider (ACG) ispreferably disposed in or near the pupil of the objective lens (O) or ina conjugate plane of the beam path.

FIG. 17 shows an example in which the subject matter of the presentinvention is combined to advantage with the concept of an achromaticbeam divider. Optical axis (OA), objective lens (O), excitation light(AL), detection light (DL), intermediate plane (ZE), periodic structure(PS), phase element (PE), achromatic beam divider (ACG).

In the following section, a practical example of an achromatic beamdivider (ACG) will be described, such as can be used in theconfiguration shown in FIG. 17. In FIG. 18, two variants a) and b) ofthe achromatic beam divider (ACG) are shown. The Figure shows atransparent substrate (TS) with reflecting regions (RB). The element(ACG) is configured in such a manner that (OA) marks the pass point ofthe optical axis through the substrate (TS), and the axis of symmetry(SA) of the element (ACG) in FIG. 17 is perpendicular to the drawingplane. The two variants a) and b) differ in that element a) reflects onthe optical axis while variant b) in the region of the optical axis doesnot reflect and has a high transmissivity. The site of the optical axisat the same time is also the position of the 0^(th) diffraction order.In variant b), the 0^(th) order is not reflected because of thetransparency at this site and therefore does not reach the specimen inthe form of excitation light. This can enhance the contrast of theillumination structure on the specimen. The achromatic beam divider(ACG) can also be constructed in such a manner that the reflectingregions along the axis of symmetry (SA) are structured and thus selectand reflect certain diffraction orders and wavelengths. If, as shown inFIG. 7, an element (ACG) is not pivotably disposed, it can only be usedto structure the illumination in one spatial direction. If the directionof the structure needs to be rotated, the element must be rotated aswell. Similarly, elements with a different orientation of the reflectivestructure that are disposed on a filter wheel could be rotated intoposition. Another option is to use an element with circular reflectiveregions in several directions as in FIG. 2. When dimensioning the sizeof the regions, the wavelength-dependent location of the diffractionorders and the location of the regions relative to the focus of theorders generated by the excitation light (AL) must be taken intoconsideration.

FIG. 18 shows the configuration of the achromatic beam divider (ACG). Atransparent substrate (TS) comprises reflecting regions (RB). The axisof symmetry (SA) passes through the pass point of the optical axis (OA).The points mark the diffraction orders, with the 0^(th) order beinglocated on the optical axis.

It should also be emphasized that the present invention, althoughpreferably used in microscopy, is not limited to the field ofmicroscopy. Because of the optical sectioning capability, this type ofconfiguration for structured illumination can also be of use in otherapplications that require 3-D imaging or scanning. Examples includesurface surveying or medical applications, such as endoscopy. Biochipreaders and high-throughput screening are other possible applications inwhich the described configurations can be used. The invention can alsobe used to advantage in microscopy and, in particular, in fluorescencemicroscopy and/or for observing living cells. In medicine, it can beused to advantage, for example, for imaging the retina of the eye(fundus imaging).

It is possible to use a number of different illumination methods andspecimen interactions. Examples are fluorescence, luminescence andreflection. The configuration for structured illumination can also becombined with the well-known TIRF (total internal reflection) method,with the structured illumination being applied to the evanescentexcitation light [12]. This can be implemented by disposing thediffraction orders (see FIG. 2) and/or the fiber configurations (FIG.13) and/or the micro-optical module (FIG. 16) in the pupil at a radialdistance so that in the specimen space, the resultant angles exceed thecritical angle of total reflection.

The light source for the configuration according to the presentinvention can be selected from a number of different designs and/orcombinations thereof: a mercury vapor lamp (HBO), laser, laser diodes,light-emitting diodes or a continuous light source (white light source).In one embodiment of the present invention, the light source can be oneof those mentioned above, without, however, being limited thereto.

One important medical use of the method for structured illuminationdescribed is fundus imaging of the eye. In this application, the methodof structured illumination can be used to advantage to reduce scatteredand background light, which can considerably enhance the contrast of theimages acquired. Furthermore, when imaging the retina of the eye, thenumerical aperture, as well as the resolution associated with thisaperture, is limited by the physiological conditions of the eye (limitedusable pupil size). In spite of the lower optical resolution, however,it is desirable to use a smaller aperture in order to reduce imagingerrors caused by the natural imperfections of the eye. Structuredillumination can therefore be used to advantage to enhance theresolution. Thus, it is possible to generate images of the retina withenhanced resolution without increasing the physical aperture of thesystem (which is difficult and entails considerable disadvantages). Thiscan lead to the desirable resolution of details of the retina (receptorrods/receptor cones) and thus offer an additional benefit to medicaldiagnostic procedures.

An important property in all applications discussed is the generation ofstructured illumination with the highest possible contrast of thestructure in the plane of the specimen. The use of coherent light in allcases allows a 100% modulation. However, when using optics systems withhigher numerical apertures, such as are normally used in microscopy,polarization plays a decisive role for the interference in the specimenplane, and thus for the contrast of structured illumination. Maximumcontrast is possible only if the polarization of the illuminating lightis perpendicular to the connecting line of the diffraction orders in thepupil plane (i.e., parallel to the location of the stripe structure inan image plane), as shown in FIG. 19. The polarization of theilluminating light must therefore either be rotated synchronously withthe rotation of the diaphragm, or azimuthally polarized light is used.The former can be generated preferably by rotating a λ/2 plate in thebeam path of the linearly polarized excitation light, with the angle ofrotation of the wave plate being half as large as that of the diaphragmstructure. Accordingly, a rotatable wave plate should be disposed in thebeam path of FIG. 11 between source (23) and main color divider (13). Inthe fiber optical configurations of FIGS. 14-16, the correctpolarization is ensured by the appropriate orientation ofpolarization-preserving fibers. Azimuthally polarized light can begenerated from linearly polarized light by means of various methodsknown from the prior art; one example is described in [11]. This type ofpolarized light makes it possible, in particular, to use illuminationwith simultaneous structuring in several spatial directions. As analternative, the rotating mask can also be fitted with a polarizerwhich, as shown in FIG. 19, transmits only linearly polarized light.This entails a rotation-dependent loss of light, see FIG. 20, which canbe compensated for by suitably synchronized light modulation.

FIG. 19 (a) shows the light distribution that is generated in the pupilby the structure shown in FIG. 1. The points marked in green are thesites of the diffraction orders, and the red ring shows the boundary ofthe pupil. The arrows identify the optimum polarization states of thelight. If the 0^(th) order contributes to the interference in thespecimen plane, this interference must have the same polarization as thehigher orders along the broken line. (b) shows the orientation of apolarizer in the mask and the preferred linear input polarization of thelight.

FIG. 20 shows the transmission through a polarizer in the mask as shownin FIG. 19 and correction of the losses by power regulation as afunction of the angle of rotation (at 0°, the axis of the polarizer andthe input polarization coincide).

While the invention has been illustrated and described in connectionwith currently preferred embodiments shown and described in detail, itis not intended to be limited to the details shown since variousmodifications and structural changes may be made without departing inany way from the spirit of the present invention. The embodiments werechosen and described in order to best explain the principles of theinvention and practical application to thereby enable a person skilledin the art to best utilize the invention and various embodiments withvarious modifications as are suited to the particular use contemplated.

REFERENCES

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1. A configuration for the optical detection of an illuminated specimen,comprising illuminating light having a light path, and a microscopeobjective lens, wherein illuminating light impinges in a spatiallystructured manner in at least one plane on said specimen, therebycreating a spatially periodic light structure in the specimen, andseveral images of the specimen are acquired by a detector in differentpositions of the light structure on the specimen, from which images anoptical sectional image and/or an image with enhanced resolution iscalculated, an illuminating-light distribution generated in or near thepupil of said objective lens or in a plane conjugate to said pupil, saidlight distribution being formed by at least two light spots, and amovable diaphragm for selecting two of said light spots, said diaphragmbeing disposed in or near the pupil of said objective lens or in a planeconjugate to said pupil, wherein said periodic light structure is formedby said objective in the specimen from said selected light spots andwherein said different positions of the light structure are created byselecting different sets of said light spots via said diaphragm.
 2. Theconfiguration of claim 1, wherein a spatially periodic structure forcreating said light spots is disposed in an intermediate image plane. 3.The configuration of claim 1, wherein the structure has an N-foldsymmetry, N=2, 4, 6 . . . , which, in or near the pupil, generates saidlight spots for structuring in N/2 spatial directions within thespecimen, which illumination patterns are symmetrical relative to theoptical axis.
 4. The configuration of claim 1, further comprising aphase plate located in the path of the illuminating light.
 5. Theconfiguration of claim 4, wherein the phase plate is circular withregions of different phase delays, which regions have at least partiallythe shape of circular sectors, and wherein said phase plate can rotateabout the optical axis.
 6. The configuration of claim 4, wherein thedifferent phase delays are produced by different refractive indicesand/or different plate thicknesses.
 7. The configuration of claim 4,wherein the phase delay for chromatic correction varies perpendicular tothe optical axis.
 8. The configuration of claim 4, wherein said phaseplate can be continuously rotated or moved and is synchronized with theimage acquisition, and wherein an image is acquired once a region with aphase delay different from the preceding one is reached.
 9. Theconfiguration of claim 4, wherein said phase plate can be rotated ormoved in discrete steps.
 10. The configuration of claim 1, wherein saiddiaphragm is a circular mask with oppositely lying transmissionopenings, which mask can rotate about the optical axis.
 11. Theconfiguration of claim 4, wherein said diaphragm and said phase plateare disposed on two rotatable or movable elements that are connected toeach other.
 12. The configuration as in claim 11, wherein the connectionis implemented via a gear unit.
 13. The configuration of claim 11wherein said diaphragm and/or phase plate is/are driven by a steppingmotor.
 14. The configuration of claim 11, wherein the light distributionis produced by outputs of fiber optics.
 15. A method for the opticaldetection of an illuminated specimen using illuminating light having alight path and a microscope objective lens, wherein the illuminatinglight impinges in a spatially structured manner in at least one plane onthe specimen, thereby creating a spatially periodic light structure inthe specimen, several images of the specimen are acquired by a detectorin different positions of the light structure on the specimen, fromwhich images an optical sectional image and/or an image with enhancedresolution is calculated, said method comprising: generating anilluminating-light distribution in or near the pupil of said objectivelens or in a plane conjugate to said pupil; said light distributionbeing formed by at least two light spots; selecting two of said lightspots via a movable diaphragm disposed in or near the pupil of saidobjective lens or in a plane conjugate to said pupil, wherein saidperiodic light structure is formed by said objective in the specimenfrom said selected light spots and wherein said different positions ofthe light structure are created by selecting different sets of saidlight spots via said diaphragm.
 16. The method of claim 15, wherein saidlight spots are created by providing a spatial periodic structure in anintermediate image plane.
 17. The method of claim 16, wherein saidstructure has an N-fold symmetry, N=2, 4, 6 . . . , which, in or nearthe pupil, generates said light spots, symmetrical relative to theoptical axis, for a structure in N/2 spatial directions within thespecimen.
 18. The method of claim 15, further comprising providing aphase plate in the path of the illuminating light.
 19. The method ofclaim 18, wherein said phase plate has a circular shape with regionswith different phase delays, which regions have an at least partiallycircular shape, and can be rotated about the optical axis.
 20. Themethod of claim 18, wherein a different phase delay is set by means ofdifferent refractive indices and different plate thicknesses.
 21. Themethod of claim 18, wherein said phase delay for the purpose ofchromatic correction is varied perpendicular to the optical axis. 22.The method of claim 18, wherein said phase plate is continuously rotatedor moved and synchronized with the image acquisition, and wherein animage is acquired once a region with a phase delay different from thepreceding one is reached.
 23. The method of claim 18, wherein said phaseplate is rotated/moved in discrete steps.
 24. The method of claim 15,wherein said light spots are selected by way of said movable diaphragmwhich is disposed in or near the pupil of the objective lens or in aplane conjugate to said pupil.
 25. The method of claim 24, wherein saiddiaphragm is a circular mask with oppositely lying transmission openingsand wherein said diaphragm rotates about the optical axis.
 26. Themethod of claim 18, wherein said diaphragm and said phase plate aredisposed on two rotatable/movable elements that are connected to eachother.
 27. The method of claim 15, wherein the light distribution isgenerated by outputs of fiber optics.